Project Summary Over 600,000 bone marrow biopsies are performed every year in the United States, while hundreds of thousands more lymph node biospies are performed. Histological evaluation of these biopsies is a critical component of care for hematologic diseases including leukemia, lymphoma, myelodysplastic syndrome, myeloproliferative disease and non-neoplastic conditions such as viral infections and autoimmune conditions. We have developed a platform that we consider a paradigm shift in the histologic examination of tissues. It is based on a new chemical process, imaging, and image processing approach that we have dubbed Clearing Histology with MultiPhoton microscopy (CHiMP).The CHiMP technology enables visual analysis of entire intact, un-embedded and uncut specimens within a short time-frame and with a resolution that is amenable to primary diagnosis. The significant clinical benefits include: 1) potential for same-day diagnosis, 2) labor and cost savings, 3) access to 3D perspective, 4) increased visual data from same specimen, 5) complete tissue preservation for ancillary studies such as DNA analysis and 6) inherent benefits of digital data such as reduced risk of loss, ready remote review by experts, and amenability to machine learning tools. Hematopoietic tissue evaluation would similarly benefit from these advantages, but unfortunately the systems developed thus far lack the resolution typically needed for visual examination of hematopoietic tissues. For this Phase I SBIR proposal, an objective is to develop customized optics to improve the resolution of our current microscopes and thereby enable use in the specialized field of hematology. Commercially available objective lenses that are compatible with our immersion medium are either limited to numerical apertures (NA) that are less than one, affecting resolution and image quality, or have insufficient working distances for imaging past the coverslip and surface roughness to obtain complete sections. We will design and test a custom objective lens with high NA and long working distance, suited for our proprietary reagents. Integrating such a lens into our microscope will also require the design of a custom scan lens, custom beam conditioning optics, and a custom polygon scanner. An associated goal is to develop a novel approach to preparing bone marrow aspiration specimens that will make them amenable to imaging with CHiMP, potentially reducing the need for core biopsies by permitting unambiguous morphologic categorization of cell subtypes in their architectural context, without the routine need for immunohistochemistry, and while preserving nucleic acids for molecular/genetic evaluation.